Zhang et al. BMC Medical Genetics(2020) 21:230
https://doi.org/10.1186/s12881-020-01150-7

RESEARCH ARTICLE

Open Access

Next generation sequencing of RB1gene forthe molecular diagnosis of ethnic minority
with retinoblastoma in Yunnan
Zhen Zhang1,2†, Yi-shuang Xiao3†, Ru Shen4†, Hong-chao Jiang1, Li Tan4, Ren-qiu Li4, Xiao-hong Yang4,
Huai-yu Gu2* , Wen-Ji He4* and Jing Ma1,5*

Abstract
Background: Retinoblastoma is a rare intraocular malignancy and typically initiated by inactivating biallelic mutationsof RB1 gene. Each year, ~ 8000 children worldwide are diagnosed for retinoblastoma. In high-income countries, patient
survival is over 95% while low-income countries is ~ 30%.If disease is diagnosed early and treated in centersspecializing in retinoblastoma, the survival might exceed 95% and many eyes could be safely treated and support alifetime of good vision. In China, approximate 1100 newly diagnosed cases are expected annually and 28 hospitals
covering 25 provinces established centers classified by expertise and resources for better treatment options and followup. Comparing with other province of eastern China, Yunnan province is remote geographically. This might result that
healthcare staff have low awareness of the role of genetic testing in management and screening in families.
Methods: The patients with retinoblastoma were selected in Yunnan. DNA from blood was used for targeted genesequencing. Then, an in-house bioinformatics pipeline was done to detect both single nucleotide variants and small
insertions/deletions. The pathogenic mutations were identified and further confirmed by conventional methods andcosegregation in families.
Results: Using our approach, targeted next generation sequencing was used to detect the mutation of these 12
probands. Bioinformatic predictions showed that nine mutations were found in our study and four were novel
pathogenic variants in these nine mutations.
Conclusions: It’s the first report to describe RB1 mutations in Yunnan children with retinoblastoma. This study would
improve role of genetic testing for management and family screening.
Keywords: Retinoblastoma, Mutations, Targeted next generation sequencing, Genetic forms* Correspondence: 2218458245@qq.com; Hewenji@etyy.cn; Jingma@etyy.cn†
Zhen Zhang, Yi-shuang Xiao and Ru Shen are co-author.
2
Department of Human Anatomy, Zhongshan School of Medicine, Sun
Yat-Sen University, Guangzhou, Guangdong 510080, PR China
4
Kunming Children’s Hospital, Kunming Medical University, Kunming, Yunnan
650228, PR China
1
Key Laboratory of Childrenʼs Major Disease Research, and Yunnan Instituteof Pediatrics, Kunming Childrenʼs Hospital, Kunming Medical University,
Kunming, Yunnan 650228, PR China
Full list of author information is available at the end of the article© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate ifchanges were made. The images or other third party material in this article are included in the article's Creative Commonslicence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to thedata made available in this article, unless otherwise stated in a credit line to the data.

Zhang et al. BMC Medical Genetics(2020) 21:230

Background
Retinoblastoma (RB, OMIM#180200) is a rare malignant
tumor which rapidly develops from the immature cellsof a retina and occurs in infancy or in children, usually
before the age of 5 years (about 2/3 children below theage of 2 years, about 95% children below the age of 5
years) [1, 2].
To explain mechanisms of RB, Knudson proposed thefamous “two-hit hypothesis”. A germline mutation (‘firsthit’, M1) and an acquired somatic mutation (‘second hit’,M2) arise heritable retinoblastomas; two somatic mutations presented in the same transformation suppressor
gene in a susceptible cell, result from non-heritable retinoblastomas [3]. In some patients, Chromosomal deletions pointed to a chromosome (chr.) 13q14 locus. In
70% of retinoblastoma tumors, the heterozygosity of chr.
13q14 polymorphic loss suggested the second hit involved the same locus [4].
The first cloned gene of tumor suppressor is retinoblastoma 1 (Rb1) gene. The protein which is encoded bythis gene is a cell cycle negative regulator. To maintainthe overall chromatin structure, this encoded protein
stabilizes constitutive heterochromatin. Defect in Rb1 is
a cause of childhood cancer Rb, osteogenic sarcoma, andbladder cancer [5].
The gene of RB1 displays a wide spectrum of mutations, including small insertions/deletions (indels), large
deletions/duplications, structural variations (SVs), andsingle nucleotide variations (SNVs) [6–8]. These mutations which are spanning 27 exons are distributed
throughout the entire length of the RB1gene. No hotspots have been reported in this gene [9].
The incidence of retinoblastoma is approximately 1 in
16,000–18,000 live births, regardless of sex, race, or geography [10, 11]. In the United States and Europe, incidence
rate of RB is 2–5 cases per million children according to
WHO [12]. In India, rates of RB incidence are 1.9–12.3
and 1.3–6.7per million in boys and girls, respectively [13].
Dimaras Helen et al. have estimated that approximate
8000 new cases are predicted each year in worldwide [4].
In China, there are about 1100–1500 new cases each year,
but only 50% those children survive [14]. The disease-freesurvival rate of children with RB in developed countries isover 95% [15] while this is substantially lower, at 10–30%
in developing countries [16, 17].
Six countries of Asia-Pacific region bear 42.6% global
burden of RB (3452 of 8099 children):1486 children in
India (18.3%), 1103 children in China (13.6%), 277 children in Indonesia (3.4%), 260 children in Pakistan(3.2%), 184 (2.3%) children in Bangladesh, 142 (1.8%)
children in Philippines [18].
A heritable form and non-heritable form are twoforms of the RB disease. Heritable RB accounts for 45%
of all cases which 80% bilateral, 15% unilateral and 5%

Page 2 of 9trilateral. Approximately 55% of cases are non-heritable
RB that is always unilateral [19]. The mode of RB inheritance is autosomal dominant (AD) found in 10% ofcases.
[20]. The majority of RB patients are sporadic and theonly affected members of otherwise unaffected families.
The disease is labeled “sporadic” if the family has no history of RB [21].
From presenting signs and clinical examination, diagnosis of RB is usually clear [22]. Leukocoria (white pupil)is the most common sign; Strabismus is the second mostcommon sign when central vision is lost. Due to increased pressure, or non-infective orbital inflammation,
advanced disease might present with enlarged cornea
and iris colour change [4, 23].
The primary goal of management is to save the child’slife, followed by salvage of the eye and optimization ofvisual function. Many treatment options of RB are available and depend on the laterality and extent of RB disease [24]. The various treatment modalities forretinoblastoma includes enucleation of the eye, externalbeam radiotherapy, brachytherapy, thermotherapy, laser
photocoagulation, cryotherapy,systemic chemotherapy,
intra-arterial chemotherapy, nanoparticulate chemotherapy and chemoreduction [4]. When children with RB
have symptoms, such as leukokoria and strabismus, it islate for these children to have best time for cure. Delayed diagnosis results in incurable invasion of the optic
nerve and brain and metastases elsewhere in the body.
However, if noticed early, prompt treatment can curethe cancer and save the eye(s) [4]. According to Intraocular International Retinoblastoma Classify (IIRC) which
divides retinoblastomas into 5 groups (labeled A throughE; Group A means small tumors while group E means
large tumor and the eye have no chance to be saved),Children’s Hospital of Fudan University reported thatabout 75% children with RB were diagnosed at group D
and E of IIRC when these children saw doctor for the
first time [25, 26].
In the management of RB, genetic testing and counseling are extremely important [27]. Heritability form of RB
can be identified by genetic testing of the proband [28].
But, genetic testing has not been carried out in Yunnan
whose permanent population was 47,368,000, including
51.48% male population and 48.52% female population,
17.79% children and 62.21% adults, 66.43%Han population and 33.57% minority population according to the
1% population sample survey by Yunnan Bureau of Statistics (http://stats.yn.gov.cn/).
Therefore, how to carry out genetic testing is an urgent problem in the RB clinical in developing countries.
In this paper, the genes of RB family in Yunnan werestudied in order to obtain genetic forms of RB by genetic
testing of the probands and then give counseling toZhang et al. BMC Medical Genetics(2020) 21:230

these families. We use targeted next generation sequencing (NGS) to screen probands and then Sanger sequencing was used to identify variants in other members of
pedigrees. In our study, nine mutations were found andfour were novel mutations.

Methods
Subjects

The non-consanguineous families from the Yun-Gui
Plateau was recruited by the Children’s Hospital of Kunming Medical University for genetic diagnosis. In thesefamilies, the children suffered from RB, but the parents
and other members were normal. This study was approved by the Ethics Committee of the Children’s Hospital of Kunming Medical University; in accordance withthe principles of the Declaration of Helsinki written informed consent was obtained from the participants ortheir guardians. The age range of the patients, in which
we obtained parental consent, is from 6 to 117 months(36.50 ± 38.61). In this study, the normal control includes 100 individuals (55 males and 45 females) aged
between 5 and 35 years old without associated hereditarydiseases.
Clinical evaluations

All clinical and physical examinations were conducted in
the Children’s Hospital of Kunming Medical University
in Kunming. CT and ultrasound imaging were performed in these probands.
Targeted NGS and variant analysis

Two-millilitre peripheral blood samples were collectedfrom the probands, siblings and their parents in tubes
containing 0.2 M EDTA. Using the QIAamp DNA blood
extraction kit (TIANGEN, Beijing, China), DNA was extracted from the venous blood of each subject. According to the manufacturer’s protocol (MyGenostics, Inc.,
Beijing, China), 3 micrograms of genomic DNA wasfragmented by Covaris 32, and the 3′ end of each DNA
fragment was A-tailed. Then, Illumina adapters were ligated to these fragments. We aimed for a 350–400 basepair product, and all samples were checked with Nanodrop 2000 or Qubit systems to determine if they represented a captured library.
Each resulting captured library was loaded on an Illumina MiSeq 2000 sequencing platform, and the sequences were determined to ensure that each sample
met the desired average sequencing coverage.
Mutation analysis

Using Bcl2Fastq software (Bcl2Fastq 2.18.0.12, Illumina,Inc.), raw image files were processed for base calling andraw data generation. In addition, to get a quality score ≥20, low-quality variations were filtered out. Then, Short

Page 3 of 9

Oligonucleotide Analysis Package (SOAP) aligner software(SOAP2.21, soap.genomics.org.cn/soapsnp.html) was used
to align the clean reads to the reference human genome(UCSC hg19, http://genome.ucsc.edu/). Polymerase chain
reaction (PCR) duplicates were removed by the Picard
programme [29, 30] .The single nucleotide polymorphisms (SNPs) were determined by the SOAPsnp
programme [31]. The reads were realigned by BurrowsWheeler Aligner (BWA) software 0.7.15, and the deletions
and insertions (InDels) were detected by Genome Analysis
Toolkit software 3.7. In addition, the identified indel SNPs
were annotated using the Exome-assistant programme(http://122.228.158.106/exomeassistant). To determine
their pathogenicity, non-synonymous variants were evaluated by four algorithms, namely, PolyPhen (http://genetics.bwh.harvard.edu/pph2/), Protein Analysis Through
Evolutionary Relationships (PANTHER, www.pantherdb.
org), Sorting Intolerant from Tolerant [SIFT, (http://sift.
jcvi.org/)] and Pathogenic Mutation Prediction (Pmut;
http://mmb.pcb.ub.es/PMut/).
Mutation validation

PCR and Sanger sequencing with an ABI3500 sequencer
were used to confirm potential causative variants in thisfamily. The sites of variation were identified to compare
the DNA sequences with the corresponding GenBank(www.ncbi.nlm.nih.gov) reference sequences. The sequences of forward and reverse primers are presented insupplementary Table 1 and were used to confirm potential causative variants in this family.
Thermocycling conditions: An initial denaturation of
95 °C for 10 min, 35 cycles of denaturation at 94 °C for
30 s., annealing at 64 °C for 30 s, extension at 72 °C for
45 s and a final extension of 72 °C for 5 min. The sequences of forward and reverse primers are in supplementary Table 1.

Results
Clinical findings

Twelve patients with RB were selected for this study oftargeted RB1 sequencing from non-consanguineous
Yunnan families. There are no pathogenic or likely
pathogenic variants were detected in three probands
with our approach while nine variants in nine families.
The clinical findings of probands

In these nine families,there are seven boys and two girls(See Fig. 1). Minority nationalities have Dai, Hani, Bai,
Hui, Yi and Han. Mean age at diagnosis is 23 ± 9.9
months for unilateral and 11 ± 8.5 months for bilateralretinoblastoma in our study, respectively. Six probands
were diagnosed with bilateral RB while three with theunilateral form. There are common sign leukocoria inprobands of families 1, 3, 4, 6, 7; in family 2, left eye ofZhang et al. BMC Medical Genetics(2020) 21:230

Page 4 of 9

Fig. 1 Pedigree of families with RB. Unaffected subjects are denoted as blank while affected subjects are represented with darkened symbols. The
arrow indicates the proband. a .Pedigree of the family 1; b. Pedigree of the family 2; c. Pedigree of the family 3; d. Pedigree of the family 4;. e.
Pedigree of the family 5; f. Pedigree of the family 6; g. Pedigree of the family 7; h. Pedigree of the family 8; i. Pedigree of the family 9

Table 1 Clinical features of patients with RB
Patient

Sex

Minority
Nationalities

Age(Months)

Onset Age (Months)

Fundus Appearance

LE

RE

LE

RE

1

M

Dai

12

6

6

RB

RB

2

M

Hani

106

14 a

17

–

RB

3

F

Bai

12

9

–

RB

–

4

F

Han

18

13

13

RB

RB

5

M

Hui

28

26

26

RB

RB

6

M

Yi

11

6

–

RB

–

7

M

Hani

60

54

–

RB

–

8

M

Han

6

5

5

RB

RB

9

M

Han

10

3

3

RB

RB

Abbreviations: M Male, LE Left eye, RE Right eye, Retinoblastoma (RB);The children of these families exhibited similar clinical features of RB. Ophthalmic
examinations showed that the child in these families are affected by RB. a Removed at 15 months

(2020) 21:230

Zhang et al. BMC Medical Genetics

the proband had been removed; the proband of 5 had
strange reflection; the proband had blurred vision andshed tears in 8 and 9 (Table 1).
The clinical findings of parents

There was no congestion in the conjunctiva, no secretions in the conjunctival sac., and no tumor in both eyes.
In both eyes (OU), the microscopy of slit lamp showedthat the cornea is smooth and transparent, the thicknessof corneal is normal, the depth of anterior chamber isnormal, cornea is clear, iris texture is clear, pupil is isometric, lens is transparent, interstitial is clear and lightemission exists.
Targeted NGS

The mean read depth of coverage for each proband sample ranged from 52 to 66X, and the average throughput
depth of the target region in each sample ranged from90 to 99X. NGS produced reads from 32.8 to 56.12 million reads and the read length from 148 to 149 bp. The
reads aligned to the human genome and mapped to thetarget region with a mean coverage from 99.8 to 99.9%.
The SNPs and Indels are reported in supplementary
Table 2.
Identification of SNVs and InDels in children with RB
patients

To detect SNVs and InDels, we analyzed blood samplesof 6 bilateral patients and 3 familial unilateral patientand identified pathogenic variants in 8 patients andlikely pathogenic variants in 1 patient. Four were noveland five were previously reported (Table 2). The
spectrum of identified mutations includes 5 SNVs (1nonsynonymous and 4 stop gain), one deletion (1 frameshift), and 3 splice site variants (splice).
Eight of them were shown to be de novo, andremaining one was inherited from one of their mothers.

Page 5 of 9

Identification of pathogenic mutation

Sanger sequencing and cosegregation further confirmed
all the pathogenic variants (See Figs. 2 and 3). We detected 9 mutations which 4 were novel and 5 were
known (Table 2). Additionally, these four novel mutations were absent in 100 normal control individuals.

Discussion
RB is the most common intraocular malignancy in childhood and presents in one or both eyes. Through presenting signs and clinical examination, diagnosis of RB isusually clear [22]. In our study, five probands had leukocoria; one proband had strange reflection; one proband’sleft eye was enucleated; two had blurred vision. Meanage at diagnosis was 23 ± 9.9 months for unilateral and
11 ± 8.5 months for bilateral. According to Gene Reviews
and AlAli et al. (Mean ageat diagnosis is 24 months forunilateral and 15 months for bilateral; mean age at diagnosis to be 27 months for unilateral and 15 months forbilateral, respectively), our mean age at diagnosis is notlater than elsewhere in the world [24]. However, there
no tumor in parents of these families.. And especially infamily 3 and 7, the visions of the parents were normal.
Genetic testing of RB1 is beneficial to provide counselling for families. In patients with RB, identification ofgene alterations improves clinical management of patient
and relatives at risk [32]. Here, we have used targeted
NGS approach for the molecular analysis of Yunnan
Children with RB, based on targeted gene enrichment
and bioinformatics pipeline. We used in-house pipeline
to successfully detect both pathogenic variants in RB patients. The average throughput depth of the target region in each sample ranged from 90-99X. These met thedesired average sequencing coverage which was ensured
to provide high quality bases for sensitive and efficientvariant detection. To detect SNVs and InDels for all thesamples, we developed an automatic in-house variant
calling pipeline as freely available tools. The pathogenic
SNVs and InDels were identified by stringent criteria,

Table 2 Variants identified by NGS in blood samples of RB chidren
No.

Locations on thechromosomes

Exon

Mutation

Pathogenic
analysis

AlleleProteinCosegregation infamily1

chr13–48,942,673-48,942,675

11

c.1061_1062delnovel

pathogenic

het

p.R355Nfs*6

De novo2

chr13–49,030,485

19

3

chr13–49,039,158

22c.1960 G > C

known

Likely pathogenic

het

p.V654L

De novoc.2236 G > T

novel

pathogenic

het

p.E746X

Heterozygous mother4

chr13–49,047,526-49,047,530

5

chr13–48,955,538

24

c.2520 + 1_2520 + 4delGTGA

novel

pathogenic

het

splicing

De novo

17c.1654 C > T

known

Pathogenic

het

p.R552X

De novo6
7

chr13–49,027,168

18c.1735 C > Tknown

pathogenic

het

p.R579X

De novo

chr13–48,881,547

2

c.264 + 5G > A

novel

pathogenic

het

splicing

De novo8

chr13–48,942,685

11

c.1072C > Tknown

pathogenic

het

p.R358X

De novo9

chr13–48,923,160

6c.607 + 1G > A

known

pathogenic

het

splicing

De novo*means termination

Zhang et al. BMC Medical Genetics(2020) 21:230

Page 6 of 9

Fig. 2 The variants of the probands. Arrows denote the mutations (the proband). Mutations of retinoblastoma identified by Sanger Sequencing infamily 1, 4, 7. a The mutation of the family1; b The mutation of the family 4; c The mutation of the family 7

and 8 the pathogenic and 1 likely pathogenic variants
were further confirmed by conventional methods andcosegregation with phenotype. Targeted mutation analysis is useful to study mutations in blood and can detect
DNA variations. The RB1 gene of seven families’
member are normal and only in family 3and 7, theunaffected mother of this family are heterozygous andthe same to the proband. Seven proband’s genes are
spontaneous mutation. Four mutations were newly
discovered mutations and never reported before. With
genetic testing, mutation profiles might be created toprecisely screen mutations of relatives or subsequent
generations in these families. Four families’ other children had no mutations by gene testing while otherfive families are one-child family.
However, no RB1 mutations were found in theremaining 25% (3/12) of cases. This could be due to thefact that there are the weakness of study, like not doing
cytogenetic studies such as chromosomal microarray(Identification of chromosome translocations or large
gross deletions) and multiplex ligation-dependent probe
amplification assays (MLPA identifies deletions or rearrangements of 1 or several exons, which account for
16% of all aberrations in RB1). This possibly results in 3
unsolved cases. The combined and cost-effective approach, such as method using a combination of direct
sequencing and MLPA, were needed to accurately detect
this 3 “negative cases” in next study.
Clearly, study of social determinants of health, suchas health seeking behavior, perceptions of medicalcare, and sociocultural issues related to cancer inheritance would inform counselling approaches that meetthe needs of families [33]. This study is helpful forthe molecular diagnosis of RB in a comprehensive in
Yunnan. These might provide molecular diagnosis to
doctor for RB management because lack of genetic
testing counseling, poor access to multidisciplinary
retinoblastoma-specific health care and socioeconomicZhang et al. BMC Medical Genetics(2020) 21:230

Page 7 of 9

Fig. 3 The variants of the probands in family 3

factors are one of the factors of higher mortality globally. For the probands with germlinemutation, themolecular diagnosis also provides counseling because
the later life of patients with heritable RB displayedthat these patients have a high life¬time risk ofdeveloping second primary malignancies, such as
osteosarcomas, melanomas and soft-tissue sarcomas[34–36].

Zhang et al. BMC Medical Genetics(2020) 21:230

However, in this cohort, the small number of patientsis not enough to establish a significant frequency reference and functional studies are necessary for assigning
pathogenicity to these novel variants.

Conclusion
Here, we reported that this approach with bioinformatics
pipeline could detect variants including novel pathogenic
variants. This comprehensive approach reduces the timeand number of assays required for the detection ofpathogenic variants by conventional methods. To thebest of our knowledge, this is the first such study using
targeted NGS approach to detect pathogenic variants in
Yunnan children with RB. Overall, targeted NGS approach is becoming more feasible in clinical settings.
Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s12881-020-01150-7.
Additional file 1: Supplementary Table 1.PCR primers for
amplification. Supplementary Table 2. Coverage level through thetarget region for patients.
Abbreviations
RB: Retinoblastoma; IIRC: Intraocular International Retinoblastoma Classify;
NGS: Next generation sequencing; SOAP: Short Oligonucleotide Analysis
Package; PCR: Polymerase chain reaction; SNPs: Single nucleotide
polymorphisms; BWA: Burrows-Wheeler Aligner; InDels: Deletions andinsertions; SIFT: Sorting Intolerant from Tolerant; SNVs: Single nucleotide
variants
Acknowledgements
We thank all subjects who participated in this study.
Authors’ contributions
Z Z, Y S X, W J H and R S conceived and designed the experiments; Z Z, R S,W J H, H C J, L T, R Q Land J M performed the experiments; Z Z, Y S X, J M,
H C J, X H Y, W J H and L T analyzed the data; Y S X, R Q Land W J H
recruited patients and collected clinical information.. YS X, R Q L,R S, H C J,
and H Y G contributed to accumulation and interpretation of clinical data. W
J H, J M H Y G and Z Z coordinated the project. All authors read andapproved the final manuscript.
Funding
This work was supported by a grant from the Joint Fund of the Yunnan
Science and Technology Department and Kunming Medical-University[grants nos. 2019FE001(− 274)], the Health and Family Planning Commissionof Kunming and Yunnan [grants nos. 2017-SW (Yang)-02 and H-2018012],
Key R & D plan of Yunnan Provincial Department of science and technology----International cooperation in science and technology (grants nos.
2018IA047), Cultivation of Reserve Talents for Young and Middle-aged Academic and Technical Leaders of Yunnan Province (2019HB102), andInnovation team of Yunnan Province for comprehensive Prevention andTreatment of Children’s hearing impairment and language diseases(2019HC026), respectively. The funding bodies played no role in the designof the study and collection, analysis, and interpretation of data and in writing
the manuscript.
Availability of data and materials
The raw sequencing data of NGS are available in the NCBI’s Sequence Read
Archive (SRA) with accession number PRJNA666751 (SRR12762344,
SRR12762345, SRR12762346, SRR12762347) https://dataview.ncbi.nlm.nih.gov/
object/PRJNA666751?reviewer=mlmsrbpj2u60r1ss0ijmkgmole”. The other

Page 8 of 9data and materials are available from the corresponding author (JM) or firstauthor (ZZ) upon reasonable request.
Ethics approval and consent to participate
This study was approved by the ethics committee of Kunming Children’s
Hospital, Kunming Medical University. Written informed consents wereobtained from all participants in this study. Moreover, written informed
consents were obtained from the parents of all participants under the age of
16. The ethics committee of Kunming Children’s Hospital, Kunming Medical
University, were acquired by our team to access the data we used in our
research.
Consent for publication
All participants have provided written informed consents to publish all
identifying images, personal details and clinical details anonymously.
Moreover, written informed consent for publication of identifying images orother personal or clinical details was obtained from the parents of any
participant under the age of 18.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Key Laboratory of Childrenʼs Major Disease Research, and Yunnan Instituteof Pediatrics, Kunming Childrenʼs Hospital, Kunming Medical University,
Kunming, Yunnan 650228, PR China. 2Department of Human Anatomy,
Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou,
Guangdong 510080, PR China. 3Department of Ophthalmology, Kunming
Children’s Hospital, Kunming Medical University, Kunming, Yunnan 650228,
PR China. 4Kunming Children’s Hospital, Kunming Medical University,
Kunming, Yunnan 650228, PR China. 5Department of Otolaryngology-Head
Neck Surgery, Kunming Children’s Hospital, Kunming Medical University,,
Kunming, Yunnan 650228, PR China.
Received: 19 January 2020 Accepted: 15 October 2020

References
1. Bornfeld N, Biewald E, Bauer S, Temming P, Lohmann D, Zeschnigk M. Theinterdisciplinary diagnosis and treatment of intraocular tumors. Dtsch
Arztebl Int. 2018;115(7):106–11.
2. Dimaras H, Khetan V, Halliday W, Orlic M, Prigoda NL, Piovesan B, Marrano P,
Corson TW, Eagle RJ, Squire JA, et al. Loss of RB1 induces non-proliferative
retinoma: increasing genomic instability correlates with progression toretinoblastoma. Hum Mol Genet. 2008;17(10):1363–72.
3. Comings DE. A general theory of carcinogenesis. Proc Natl Acad Sci U S A.
1973;70(12):3324–8.
4. Dimaras H, Corson TW, Cobrinik D, White A, Zhao J, Munier FL, Abramson
DH, Shields CL, Chantada GL, Njuguna F, et al. Retinoblastoma. Nat Rev Dis
Primers. 2015;1(1):15021..
5. Lohmann D. Retinoblastoma. Adv Exp Med Biol. 2010;685:220–7.
6. Valverde JR, Alonso J, Palacios I, Pestana A. RB1 gene mutation up-date, ameta-analysis based on 932 reported mutations available in a searchable
database. BMC Genet. 2005;6:53.
7. Devarajan B, Prakash L, Kannan TR, Abraham AA, Kim U, Muthukkaruppan V,
Vanniarajan A. Targeted next generation sequencing of RB1 gene for themolecular diagnosis of retinoblastoma. BMC Cancer. 2015;15:320.
8. Lohmann DR. RB1 gene mutations in retinoblastoma. Hum Mutat. 1999;
14(4):283–8.
9. Jaya Singh AMAJ. Next-generation sequencing-based method showsincreased mutation detection sensitivity in an Indian retinoblastoma cohort.
Mol Vis. 2016;22:1036–47.
10. Kivela T. The epidemiological challenge of the most frequent eye cancer:
retinoblastoma, an issue of birth and death. Br J Ophthalmol. 2009;93(9):1129–31..
11. Broaddus E, Topham A, Singh AD. Incidence of retinoblastoma in the USA:1975-2004. Br J Ophthalmol. 2009;93(1):21–3.
12. Retinoblastoma union for International Cancer Control WHO.2014.
13. Satyanarayana L, Asthana S, Labani SP. Childhood cancer incidence in India:
a review of population-based cancer registries. Indian Pediatr. 2014;51(3):218–20.

Zhang et al. BMC Medical Genetics(2020) 21:230

14. Song Z. Effect and mechanism of silencing SHH gene on the proliferation
and apoptosis of retinoblastoma cell: Jilin University; 2016..
15. Canadian Retinoblastoma Society. National Retinoblastoma Strategy
Canadian Guidelines for Care: Strategie therapeutique du retinoblastome
guide clinique canadien. Can J Ophthalmol. 2009;44(Suppl 2):S1–S88.
16. Canturk S, Qaddoumi I, Khetan V, Ma Z, Furmanchuk A, Antoneli CB, Sultan
I, Kebudi R, Sharma T, Rodriguez-Galindo C, et al. Survival of retinoblastoma
in less-developed countries impact of socioeconomic and health-related
indicators. Br J Ophthalmol. 2010;94(11):1432–6.
17. Dean M, Bendfeldt G, Lou H, Giron V, Garrido C, Valverde P, Barnoya M,
Castellanos M, Jimenez-Morales S, Luna-Fineman S. Increased incidence anddisparity of diagnosis of retinoblastoma patients in Guatemala. Cancer Lett.
2014;351(1):59–63.
18. Jain M, Rojanaporn D, Chawla B, Sundar G, Gopal L, Khetan V.
Retinoblastoma in Asia. Eye (Lond). 2019;33(1):87–96.
19. Soliman SE, Racher H, Zhang C, MacDonald H, Gallie BL. Genetics andmolecular diagnostics in retinoblastoma--an update. Asia Pac J Ophthalmol(Phila). 2017;6(2):197–207.
20. Vogel F. Genetics of retinoblastoma. Hum Genet. 1979;52(1):1–54.
21. Yun J, Li Y, Xu CT, Pan BR. Epidemiology and Rb1 gene of retinoblastoma.
Int J Ophthalmol. 2011;4(1):103–9.
22. Dimaras H, Kimani K, Dimba EA, Gronsdahl P, White A, Chan HS, Gallie BL.
Retinoblastoma. Lancet. 2012;379(9824):1436–46.
23. Dimaras H. Retinoblastoma genetics in India: from research to
implementation. Indian J Ophthalmol. 2015;63(3):219–26.
24. AlAli A, Kletke S, Gallie B, Lam WC. Retinoblastoma for pediatric
ophthalmologists. Asia Pac J Ophthalmol (Phila). 2018;7(3):160–8..
25. Linn MA. Intraocular retinoblastoma: the case for a new group classification.
Ophthalmol Clin N Am. 2005;18(1):41–53.
26. Gao YJ, Qian J, Yue H, Yuan YF, Xue K, Yao YQ. Clinical characteristics andtreatment outcome of children with intraocular retinoblastoma: a reportfrom a Chinese cooperative group. Pediatr Blood Cancer. 2011;57(7):1113–6.
27. Skalet AH, Gombos DS, Gallie BL, Kim JW, Shields CL, Marr BP, Plon SE,
Chevez-Barrios P. Screening children at risk for retinoblastoma: consensus
report from the American Association of Ophthalmic Oncologists andPathologists. Ophthalmology. 2018;125(3):453–8.
28. Roelofs K, Shaikh F, Astle W, Gallie BL, Soliman SE. Incidental neuroblastoma
with bilateral retinoblastoma: what are the chances? Ophthalmic Genet.
2018;39(3):410–3.
29. Etherington GJ, Ramirez-Gonzalez RH, MacLean D. bio-samtools 2: a
package for analysis and visualization of sequence and alignment data withSAMtools in ruby. Bioinformatics. 2015;31(15):2565–7.
30. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis
G, Durbin R. The sequence alignment/map format and SAMtools.
Bioinformatics. 2009;25(16):2078–9.
31. Li R, Yu C, Li Y, Lam TW, Yiu SM, Kristiansen K, Wang J. SOAP2: an improved
ultrafast tool for short read alignment. Bioinformatics. 2009;25(15):1966–7.
32. Richter S, Vandezande K, Chen N, Zhang K, Sutherland J, Anderson J, Han L,
Panton R, Branco P, Gallie B. Sensitive and efficient detection of RB1 genemutations enhances Care for Families with retinoblastoma. Am J Hum
Genet. 2003;72(2):253–69.
33. He LQ, Njambi L, Nyamori JM, Nyenze EM, Kimani K, Matende I, Rono H,
Njom V, Bett J, Mukuria M, et al. Developing clinical cancer genetics services
in resource-limited countries: the case of retinoblastoma in Kenya. Public
Health Genom. 2014;17(4):221–7.
34. Kleinerman RA, Tucker MA, Abramson DH, Seddon JM, Tarone RE, Fraumeni
JJ. Risk of soft tissue sarcomas by individual subtype in survivors ofhereditary retinoblastoma. J Natl Cancer Inst. 2007;99(1):24–31.
35. Kleinerman RA, Yu CL, Little MP, Li Y, Abramson D, Seddon J, Tucker MA.
Variation of second cancer risk by family history of retinoblastoma among
long-term survivors. J Clin Oncol. 2012;30(9):950–7.
36. MacCarthy A, Bayne AM, Brownbill PA, Bunch KJ, Diggens NL, Draper GJ,
Hawkins MM, Jenkinson HC, Kingston JE, Stiller CA, et al. Second andsubsequent tumours among 1927 retinoblastoma patients diagnosed in
Britain 1951-2004. Br J Cancer. 2013;108(12):2455–63.

Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.

Page 9 of 9